Meiotic defects in a man with non-obstructive azoospermia: case report

Infertile men have an increased frequency of aneuploid sperm. We have determined that decreased recombination is associated with the production of aneuploid sperm in humans. The aim of this study was to determine whether some cases of infertility are associated with decreased meiotic recombination. Analysis of the early stages of meiosis was performed in a 33-year-old man with non-obstructive azoospermia. Newly developed immunocytogenetic techniques were used to identify the synaptonemal complex (SC) in various stages of prophase. Antibodies to meiotic proteins identified the SC (SYN1/SCP3), the centromere (CREST) and recombination sites (MLH1). Only 36 meiotic spreads were recovered from the infertile man, compared with hundreds available from controls. One-third of the cells were in zygotene compared with 4% in controls, demonstrating an inability of bivalents to synapse and progress to pachytene. The infertile man had a greatly reduced frequency of recombination, with a mean of only 32.7 MLH1 foci/cell (range 1-60) compared with 46.0 (range 21-62) in control donors. A high proportion of cells (73%) contained at least one autosomal bivalent with zero MLH1 foci, compared with only 4.5% in control donors. Discontinuities in the SC were also more prevalent (68% of cells versus 26% in controls). This is the first demonstration of dramatic pachytene-stage abnormalities in an infertile man using these powerful new immunocytogenetic techniques.     

Leukocyte detection in human semen using flow cytometry

This study set out to establish a new method, using flow cytometry, to evaluate leukocytes in semen. Ejaculates of 59 males, asymptomatic for genitourinary infections, were examined. Routine semen analyses were carried out as well as peroxidase and polymorphonuclear granulocyte-elastase detection. Leukocytes were detected combining flow cytometry and monoclonal antibodies (anti-CD45, anti-CD53). This technique reliably assessed the total number of leukocytes and differentiated subpopulations even at low concentrations. The peroxidase test and elastase determination showed good specificity, but only moderate sensitivity versus flow cytometry combined with monoclonal antibodies. No significant association was observed between semen parameters and leukocytospermia whether evaluated by conventional methods or flow cytometry except for a moderate correlation between spermatozoa and CD53-positive cell concentrations. A first comparison of data from patients grouped on the basis of leukocytospermia (>10(6) white blood cells, WBC/ml) or non-leukocytospermia revealed no significant differences in semen parameters; lowering the threshold value for leukocytospermia to 2x10(5) WBC/ml, sperm concentration was reduced in the group with a low number of WBC identified by monoclonal antibodies. Flow cytometry using monoclonal antibodies was seen to be a simple, reproducible method that enables leukocytes in semen to be accurately detected and to identify WBC subpopulations without preliminary purification procedures.     

Seminal haploid cell detection by flow cytometry in non-obstructive azoospermia: a good predictive parameter for testicular sperm extraction

BACKGROUND: Testicular sperm extraction (TESE) associated with ICSI gives patients suffering from non-obstructive azoospermia (NOA) the possibility of becoming a father. The success rate of TESE based on sperm recovery is approximately 50%, and the commonly used non-invasive parameters are not predictive enough. Only the invasive testis biopsy has a good prognostic value. The aim of this study was to evaluate the prognostic value of the detection of seminal haploid cells by flow cytometry (FCM) in order to avoid unnecessary testicular biopsy. METHODS: For 37 NOA patients undergoing testicular biopsy, we measured testis size, serum FSH and inhibin B levels and carried out seminal cytology, seminal FCM analysis and histological examination. RESULTS: Sperm were found in 18 biopsies. These results were correlated with cytology, FCM analysis and the histological examination. FCM was more sensitive than cytology (100 versus 59%) but less specific (67 versus 83.5%) whereas the histological observation of complete spermatogenesis appeared to be less sensitive (50%) but more specific (100%). CONCLUSION: Detection of seminal haploid cells by FCM appears to be an interesting non-invasive technique which can predict TESE results and improve the management of NOA patients.     

AZFb deletions predict the absence of spermatozoa with testicular sperm extraction: preliminary report of a prognostic genetic test

Genetic abnormalities, including partial deletions of the Y-chromosome,are commonly detectable in men with non-obstructive azoospermia(NOA). NOA can be treated using testicular sperm extraction(TESE) with intracytoplasmic sperm injection (ICSI). Recentstudies have shown that the presence of deletions involvingthe AZFc region do not appear to affect the chance of retrievingspermatozoa or have a significant impact on fertilization orpregnancy rates with ICSI. We investigated the effect of Y-chromosomepartial deletions on the chance of sperm retrieval with TESE.Eighty attempts at sperm retrieval were performed using TESEon men who were previously evaluated for Y-chromosome partialdeletions. Y-chromosome analysis was performed using a polymerasechain reaction (PCR)-based technique with 35 sequence-tagged-sites.Of the 80 men, nine (11%) had partial Y-chromosome deletionsdetected. Two azoospermic men with AZFc deletions had successfulsperm retrieval, ICSI and a subsequent clinical pregnancy. Sevenmen had deletions involving the AZFb region (three men had isolatedAZFb deletions, one had AZFa, AZFb and AZFc deleted, and threehad AZFb and AZFc deleted). None of the seven men had spermatozoaextracted by TESE, a result that is significantly differentfrom the overall 64% (47/73) sperm retrieval rate achieved atour centre (P = 0.001). Two men with AZFb deletions had cellsconsistent with round spermatids identified and injected intooocytes without effecting any normal fertilizations. Althoughpreliminary, these results suggest that the presence of an AZFbdeletion is a significantly adverse prognostic finding for TESE.Men with AZFb deletions should be apprised of these resultsbefore attempting TESE-ICSI. Alternatives such as donor inseminationor adoption should be considered or therapy delayed until improvedresults with round spermatid injections are likely.     

Conventional multiple or microdissection testicular sperm extraction: a comparative study

BACKGROUND: Testicular sperm extraction (TESE) with ICSI is becoming the first-line treatment for non-obstructive azoospermia (NOA). Recently, the sperm retrieval rate (SRR) by microdissection TESE was reported to be higher than by conventional TESE. However, a comprehensive comparison between multiple and microdissection TESE patients including histological findings has not been reported. METHODS: Patients with NOA who underwent microdissection TESE (n = 56) or multiple TESE (n = 37) were compared. Pre-operative characteristics were similar between groups. In addition, microscopic findings during microdissection TESE also were investigated. RESULTS: Operative time was significantly longer for microdissection TESE than for multiple TESE. Histological examination suggested that spermatogenesis was relatively more impaired in the microdissection TESE group than in the multiple TESE group. Despite this, SRR by microdissection TESE (42.9%) appeared higher than by conventional TESE (35.1%) although this observation failed to reach statistical significance. Seventeen of 26 patients (65.4%) with heterogeneous tubule were successful for sperm retrieval. No severe operative complications occurred in any patient in either group, and no patient required post-operative hormone replacement to treat hypogonadism. CONCLUSIONS: Microsurgical technique is safe and may improve SRR for TESE in a variety of patients with NOA, especially patients with heterogeneous testicular tubules.     

Manifestation of Y-chromosomal deletions in the human testis: a morphometrical and immunohistochemical evaluation

BACKGROUND: Deletions of the AZF (azoospermia factor) subregions on the Y chromosome are accompanied by a diverse spectrum of spermatogenic disturbances ranging from hypospermatogenesis to total depletion of germ cells causing infertility. The AZF region encodes gene products which are candidates for the genetic control of spermatogenesis. Although it is known which genes are involved, a general principle of cause and effect cannot yet be deciphered and the deletion type has non-uniform histological phenotypes. METHODS AND RESULTS: We analysed morphological parameters of testicular biopsies from 17 patients diagnosed for Y chromosome microdeletions. As control groups we analysed testes from patients with idiopathic Sertoli cell-only (SCO) syndrome (n = 11), mixed atrophy (n = 10) and complete spermatogenesis (n = 11). A detailed genetic analysis on the extension of the observed microdeletions revealed similar breakpoints in the distal and proximal region of the AZFc region, indicating a common mechanism of homologous recombination for such deletions, as has been suggested before. Morphometric parameters such as the diameter of the tubules, lumen, thickness of the lamina propria and height of the tubule epithelia were investigated. The diameter of the tubules from patients with microdeletions was found to be significantly smaller compared with patients with mixed atrophy. Considering also the size of the tubules, lumen and epithelia, a Y-chromosomal microdeletion represents an intermediate state between an idiopathic SCO and normal spermatogenesis. The immunohistochemical analysis of six different Sertoli cell markers, cytokeratin 18, vimentin, inhibin alpha subunit, 14-3-3 theta, FSH receptor and androgen receptor, revealed no impact of AZF deletion on the specific expression pattern of these genes. CONCLUSIONS: Our results suggest that, notwithstanding the deletion of a common region in the AZFc region, microdeletions of the Y chromosome lead to an intermediate status between idiopathic SCO and complete spermatogenesis, resulting in a heterogeneous histological profile regardless of the seminiferous activity. The Sertoli cell function seems not to be altered.     

The highly conserved NANOS2 protein: testis-specific expression and significance for the human male reproduction

The highly conserved Nanos gene was found to encode a translationalrepressor necessary for germ-cell development in lower organisms.The mammalian homologue, Nanos2, was recently found to be expressedin the mouse germ cells. Since its disruption caused infertilityexclusively in males, we sought to study the significance ofthis gene in human male reproduction. Here, we describe forthe first time the expression pattern of the NANOS2 gene inhuman tissues and show that it is testis specific. We foundthat NANOS2 protein is present in prenatal germ cells and atlater stages in spermatogenesis. To elucidate the role of NANOS2in human germ-line development, we screened this gene for mutationsin 214 males with isolated sterility and spermatogenic abnormalities.We identified two heterozygous variants, each in a differentoligospermic patient, the second allele being the wild-type.The influence of the first variant, a missense mutation H68Qon the sterility phenotype, was not obvious since it was accompaniedby a microdeletion within the AZF region of the Y chromosome.The second variant contained a silent mutation, H109H. Althoughboth mutations were situated within the most conserved RNA-bindingdomain and were absent in 400 fertile males, it is not obviousthat they cause male infertility.     

Testicular fine needle aspiration: the alternative method for sperm retrieval in non-obstructive azoospermia.

The objective of this prospective open study was to determine the feasibility of obtaining mature spermatozoa for intracytoplasmic sperm injection (ICSI) by testicular fine needle aspiration (TEFNA) in men diagnosed with non-obstructive azoospermia. TEFNA consisted of a mean of 15 punctures and aspirations in each testis, using 23 gauge butterfly needles, connected to a 20 ml syringe with an aspiration handle. Patients (n = 85) underwent 111 TEFNA cycles. Mature testicular spermatozoa were recovered in 65 (58.5%) cycles from 50 (58.8%) patients. The sperm recovery rate by testicular histology was 14 out of 29 (48.3%) in patients with Sertoli cell-only, 13 out of 28 (46.4%) in patients with maturation arrest, 19 out of 20 (95%) in patients with hypospermatogenesis, four out of six (66.6%) in patients with tubular hyalinization due to non-mosaic Klinefelter's syndrome. No spermatozoa were found in two cases with post-irradiation fibrosis. ICSI was performed in all 65 cycles. In 58 cycles in which only the husbands' spermatozoa were used, 406 mature oocytes were injected, and 154 (37.9%) were normally fertilized. Of the 143 embryos that developed (92.8%), 119 were transferred in 42 cycles resulting in 18 clinical pregnancies (42. 8%), with 31 gestational sacs, providing an implantation rate of 26%. One abortion of a singleton pregnancy occurred (5.6%). No major side-effects, such as haematoma or infection were recorded. In conclusion, we have found TEFNA to be efficient, easy to learn, safe and well tolerated by all patients. In our opinion, TEFNA should be considered the first choice whenever sperm recovery is attempted in patients with non-obstructive azoospermia.     

A new multiparameter flow cytometric method for human semen analysis

BACKGROUND: The objectives of this study were (i) to evaluate whether the combined use of Syto 16 and 7-amino-actinomycin-D (7-AAD) allows the detection of sperm apoptosis and (ii) to describe a new multiparameter flow cytometric method to assess simultaneously sperm concentration (SC), viability and apoptosis as well as leukocyte concentration. METHODS: Semen samples from 68 patients were evaluated according to World Health Organization (WHO) criteria (normal, n=26; abnormal, n=42). The detection of activated caspases before and after betulinic acid (BA) incubation was carried out in 13 semen samples by flow cytometry using fluorescein-labelled inhibitors of caspases (FLICA). A multiparameter flow cytometric analysis was performed in 55 semen samples. Fluorescent microspheres were used to assess SC. Sperm apoptosis was detected by staining sperm with Syto 16 and 7-AAD. Leukocytes were counted using monoclonal anti-CD45. RESULTS: A significant correlation between the percentage of the spermatozoa with low Syto 16 fluorescence and the percentage of spermatozoa containing activated caspases was found (r=0.68, P=0.0106; n=13). After incubation with BA, an increase of the percentage of apoptotic cells was observed in all samples, using both the Syto 16/7-AAD and the caspase activation methods. There was a good correlation between flow cytometry and optical microscopy for sperm (r=0.98, P < 0.0001) and leukocyte counting (r=0.64, P <0.0001). The percentage of apoptotic sperm was inversely correlated with both SC (r=-0.303, P=0.0246) and morphology (r=-0.384, P=0.0050) but not with motility. CONCLUSIONS: The combination of Syto 16/7-AAD provides a sensitive assay to detect sperm apoptosis. The multiparameter flow cytometric method described offers the possibility of a simultaneous, simple, rapid and accurate assessment of several semen parameters.     

Factors influencing the outcome of ICSI in patients with obstructive and non-obstructive azoospermia: a comparative study

BACKGROUND: Factors influencing success of sperm retrieval in azoospermic patients and outcome of ICSI were evaluated. METHODS AND RESULTS: Uni- and multifactorial analysis were performed using logistic and stepwise analysis, following surgical sperm retrieval by percutaneous epididymal sperm aspiration (55 cycles) or testicular sperm extraction (142 cycles) in 52 and 123 patients with obstructive azoospermia (OA) and non-obstructive azoospermia (NOA) respectively. ICSI cycles using fresh or cryopreserved-thawed sperm were included. Sperm were retrieved to allow ICSI in 100 and 41% of OA and NOA patients, with no significant correlation with patients' age or FSH level. Occurrence of pregnancy was significantly correlated with female age (90th quantile: 38 years), number of oocytes retrieved (10th quantile: five oocytes) and number of oocytes injected (10th quantile: four oocytes). Sperm origin (epididymal versus testicular), status (fresh or thawed), male partner's age, and serum FSH had no significant effect upon implantation rate, pregnancy rate per embryo transfer or spontaneous miscarriage rate. CONCLUSIONS: In OA patients ICSI should be planned in conjunction with surgical sperm retrieval. In contrast, the lack of efficient non-invasive parameters to predict sperm retrieval in NOA suggests that elective surgical sperm retrieval may be offered to these patients prior to ovarian stimulation of their partners, especially when donor back-up is not an alternative. Female factors such as age and ovarian reserve have significant impact upon clinical success rates.     

Cytogenetic and molecular characterization of the derivative Y chromosome: a case study of an azoospermic patient

The success of infertility treatment depends on the underlying cause and severity of the infertility problem. The current report addresses the complex genotype–phenotype interactions in an azoospermic man. Cytogenetic, molecular cytogenetic and molecular genetic studies indicated the derivative monocentric Y chromosome with duplication of Yp11 (including SRY gene) and partial deletion of Yq11 (including azoospermia factor – AZFb-c regions) as the most probable cause of the severe testicular failure. Our study emphasizes the importance of detailed genetic analysis in male infertility evaluation and helps to estimate the outcome of infertility treatment.     

Toluidine blue cytometry test for sperm DNA conformation: comparison with the flow cytometric sperm chromatin structure and TUNEL assays

BACKGROUND: Sperm DNA integrity (SDI) is an important factor in the prognosis of male fertility. Here we compare the toluidine blue (TB) image cytometry test, recently proposed by us for SDI assessment, with two other tests-the sperm chromatin structure assay (SCSA) and the terminal nick-end labelling (TUNEL) assay. METHODS: Sperm samples from 35 men were evaluated for standard sperm parameters and subjected to the TB test and SCSA. Eighteen of the 35 samples were also subjected to the TUNEL assay. RESULTS: The proportion of sperm cells with abnormal DNA integrity assayed by the TB test correlated strongly with the proportion of abnormal cells detected by the SCSA and TUNEL assay (rho=-0.84 and rho=0.80, P<0.001, respectively). Furthermore, the fractions of abnormal cells by the TB test corresponded closely to the sum of two SCSA parameters, the DNA fragmentation index (DFI) and the fraction of highly DNA-stainable cells (HDS) (medians 33.0 versus 32.0%, P=0.6). CONCLUSIONS: Abnormal cells in a TB test correspond to the sum of DFI and HDS fractions in the SCSA. TB-positive cells may represent sperm with fragmented DNA and/or abnormal chromatin structure. Because the TB test is an easy and inexpensive method, its potential use as a routine test for sperm DNA integrity, complementary to standard semen parameters, should be investigated further.     

Spermatid injection into human oocytes. II.Clinical application in the treatment of infertility due to non obstructive azoospermia

We have reported recently the first birth after intrauterinetransfer of embryos obtained by injection of round spermatidsinto oocytes in cases of unexpected azoospermia. Here we providea complete documentation of the series of 11 cases in whichthis novel method of infertility treatment was employed. Infour of these cases, elongated spermatids were identified inthe ejaculate, and it was decided to perform elongated spermatidinjection (ELSI). In the other six cases, only round spermatidswere present, and round spermatid injection (ROSI) was done.In one case, ROSI was given preference to ELSI because of avery poor viability status of elongated spermatids present inthe ejaculate. Fertilization of at least one oocyte was achievedin 10 of the 11 treatment cycles; the fertilization rate inthese 10 cycles ranged between 7 and 100% with a mean valueof 45%. All of the two-pronucleated zygotes cleaved and weretransferred to the patient’s uterus. A singleton pregnancywas achieved in two ROSI cycles. Both pregnancies developeduneventfully and resulted in the birth of normal infants. Thesedata show that intra-ooplasmic injection of spermatids obtainedfrom the ejaculate may become the treatment of first choicein patients with non-obstructive azoospermia.     

Andrology: Clinical and biological characteristics of infertile men with a history of cryptorchidism

Out of 85 fertile and 1014 infertile men, two (2.4%) and 95(9.4%) respectively had a history of cryptorchidism. Thus cryptorchidismappears to be a risk factor for fertility since this differencewas significant. Further comparisons showed that the volumeof a former cryptorchid testis was smaller than the contralateralnormally descended one and that sperm output/concentration wasmore impaired in bilateral than in unilateral cryptorchidism.A retractile testis, defined as a testis reported by the patientto be spontaneously and regularly, i.e. at least once a week,ascending up into a supra-scrotal position, was more frequentin infertile men with a history of cryptorchidism than in fertilemen. Retractility was more frequent on the cryptorchid side,and was found more frequently after hormonal than after surgicaltreatment. Independently of all epidemiological and clinicalparameters studied, retractility was associated with a lowersperm output. Among the infertile men with a history of cryptorchidism,45% had an abnormally high scrotal temperature. This abnormaltemperature represented a pejorative risk factor for fertilityin this group, since it was associated with a more severelyimpaired spermatogenesis and a higher incidence of primary infertilitythan in infertile men with a history of cryptorchidism but normalscrotal temperatures.     

A two-step protocol for the detection of rearrangements at the AZFc region on the human Y chromosome

The AZFc region on the human Y chromosome consists mainly of very long direct and inverted repeats and is prone to rearrangement. Although deletion of the entire AZFc is found only in subfertile men, duplications and deletions of portions of AZFc as well as inversions are quite common and represent major polymorphisms of the Y chromosome. Several methods are available to detect these rearrangements, and each has its own advantages and limits. We designed a two-step PCR protocol to study the polymorphic structure of AZFc. The first PCR determines the copy number of the Deleted in Azoospermia (DAZ) genes within AZFc using the autosomal DAZ-Like gene as a dosage control, and the results could be verified by dosage Southern blot analyses. The second PCR simultaneously detects five sequence tagged sites (STSs) that are either present or absent in the various AZFc partial deletions. One of the STSs, sY1291, was found to be polymorphic in size due to varying lengths of a poly-T stretch. A combination of the DAZ dosage PCR and the 5-STS multiplex PCR reaction detects most, if not all, deletions and duplications at AZFc. It offers a simple and reliable way to screen large populations for AZFc rearrangements and study their effects on male fertility.     

Assisted reproduction with in-vitro-cultured testicular spermatozoa in cases of severe germ cell apoptosis: a pilot study.

BACKGROUND: Apoptosis-related cell damage is known to compromise success rates of assisted reproduction with ejaculated spermatozoa. This study was undertaken to determine whether the frequency of apoptosis-related cell damage and reproductive performance of testicular spermatozoa from men with non-obstructive azoospermia can be improved by in-vitro culture. METHODS: Testicular tissue samples were cultured for 2 days in the presence of 50 IU/l FSH and 1 micromol/l testosterone. The frequency of spermatozoa showing DNA strand breakage and plasma membrane phosphatidylserine externalization was compared in before-culture and after-culture samples. The after-culture samples were used in assisted reproduction attempts. RESULTS: In a group of 11 azoospermic patients with at least two previous intracytoplasmic sperm injection (ICSI) failures, the incidence of DNA strand breakage was high in living testicular spermatozoa from before-culture samples, but significantly lower in after-culture samples (96 versus 30%, P < 0.001). The same applied to the incidence of phosphatidylserine externalization in the motile sperm subpopulation from the before-culture and after-culture samples (83 versus 6%, P < 0.001). Seven ongoing clinical pregnancies (six with fresh embryos and one with cryopreserved embryos) were established. CONCLUSIONS: Severe testicular sperm apoptosis may become a new indication for testicular tissue in-vitro culture before ICSI.     

In vivo expression of rubella antigens on human leucocytes: detection by flow cytometry

Flow cytometry has been used to detect in vivo expression of rubella antigens on human leucocytes. Sequential samples of peripheral blood were obtained from four volunteers with naturally acquired rubella and five persons immunised with RA27/3 rubella vaccine. Leucocytes were stained for rubella antigens using a pool of rubella monoclonal antibodies. Rubella antigens were detected on the leucocytes of all four volunteers with naturally acquired rubella between 1 and 13 days after onset of illness. Viral antigens were expressed more frequently on the monocyte (9-51%) than the lymphocyte (less than 1-4%) and granulocyte (less than 1-3%) populations. Among the vaccines, rubella antigens were detected on the leucocytes of four of the five volunteers between 5 and 12 days after immunisation. The expression of viral antigens was more transient and the proportion of cells exhibiting rubella-specific fluorescence considerably lower following vaccination (1-12%) than natural infection (9-51%). Our results demonstrate that flow cytometry provides a rapid and sensitive analytical technique for detecting viral antigens on leucocytes from infected persons. Leucocytes may play an important role in the pathogenesis of rubella infection.     

The use of cryopreserved aged human oocytes in a test of the fertilizing capacity of human spermatozoa.

The use of cryopreserved aged human oocytes in a diagnostic test of sperm fertilizing ability was evaluated. Oocytes arising from assisted conception cycles and showing no signs of fertilization 48 h post-insemination were cryopreserved by one of two methods. An ultrarapid method using dimethyl sulphoxide gave poor post-thaw results, with only 5/69 (7.2%) oocytes surviving. Oocytes frozen by a slow method using propanediol as the cryoprotectant gave better survival rates (359/594; 60%). Fertilization by donor spermatozoa of these thawed oocytes was poor (15/63; 24%) when the zona pellucida was left intact. To improve this, the zona was enzymatically removed using pronase. These zona-free oocytes were then inseminated with spermatozoa from a fertile donor or from men previously exhibiting fertilization failure in an in-vitro fertilization treatment cycle. The fertilization rate in the patient group (41/91; 45%) was significantly lower than in the donor group (16/18; 89%) (P less than 0.02). There was also a significant (P less than 0.03) reduction in the median number of pronuclei per oocyte (2.9 versus 4.5). These results show that aged oocytes can be effectively cryopreserved to establish a bank for use in a test to identify men with impaired sperm fertilizing capacity.